BCMA (B-cell maturation antigen, TNFRSF17, CD269) is a transmembrane protein belonging to the TNF receptor super family. BCMA is originally reported as an integral membrane protein in the Golgi apparatus of human mature B lymphocytes, i.e., as an intracellular protein (Gras et al., (1995) International Immunol 7(7):1093-1105) showing that BCMA seems to have an important role during B-cell development and homeostasis. The finding of Gras et al. might be associated with the fact that the BCMA protein that was described in Gras et al. is, because of a chromosomal translocation, a fusion protein between BCMA and IL-2. Meanwhile BCMA is, however, established to be a B-cell marker that is essential for B-cell development and homeostasis (Schliemann et al., (2001) Science 293 (5537):2111-2114) due to its presumably essential interaction with its ligands BAFF (B cell activating factor), also designated as TALL-1 or TNFSF13B, and APRIL (A proliferation-inducing ligand).
BCMA expression is restricted to the B-cell lineage and mainly present on plasma cells and plasmablasts and to some extent on memory B-cells, but virtually absent on peripheral and naive B-cells. BCMA is also expressed on multiple myeloma (MM) cells. Together with its family members transmembrane activator and cyclophylin ligand interactor (TACI) and B cell activation factor of TNF family receptor (BAFF-R), BCMA regulates different aspects of humoral immunity, B-cell development and homeostasis. Expression of BCMA appears rather late in B-cell differentiation and contributes to the long term survival of plasmablasts and plasma cells in the bone marrow. Targeted deletion of the BCMA gene in mice does not affect the generation of mature B-cells, the quality and magnitude of humoral immune responses, formation of germinal center and the generation of short-lived plasma cells. However, such mice have significantly reduced numbers of long-lived plasma cells in the bone marrow, indicating the importance of BCMA for their survival (O'Connor et al., 2004).
In line with this finding, BCMA also supports growth and survival of multiple myeloma (MM) cells. Novak et al found that MM cell lines and freshly isolated MM cells express BCMA and TACI protein on their cell surfaces and have variable expression of BAFF-R protein on their cell surface (Novak et al., (2004) Blood 103(2):689-694).
Multiple myeloma (MM) is the second most common hematological malignancy and constitutes 2% of all cancer deaths. MM is a heterogenous disease and caused by mostly by chromosome translocations inter alia t(11; 14), t(4; 14), t(8; 14), del(13), del(17) (Drach et al., (1998) Blood 92(3):802-809; Gertz et al., (2005) Blood 106(8):2837-2840; Facon et al., (2001) Blood 97(6):1566-1571). MM-affected patients may experience a variety of disease-related symptoms due to, bone marrow infiltration, bone destruction, renal failure, immunodeficiency, and the psychosocial burden of a cancer diagnosis. As of 2006, the 5-year relative survival rate for MM was approximately 34% highlighting that MM is a difficult-to-treat disease where there are currently no curative options.
Exciting new therapies such as chemotherapy and stem cell transplantation approaches are becoming available and have improved survival rates but often bring unwanted side effects, and thus MM remains still incurable (Lee et al., (2004) J Natl Compr Canc Netw 8 (4): 379-383). To date, the two most frequently used treatment options for patients with multiple myeloma are combinations of steroids, thalidomide, lenalidomide, bortezomib or various cytotoxic agents, and for younger patients high dose chemotherapy concepts with autologous stem cell transplantation.
Most transplants are of the autologous type, i.e. using the patient's own cells. Such transplants, although not curative, have been shown to prolong life in selected patients. They can be performed as initial therapy in newly diagnosed patients or at the time of relapse. Sometimes, in selected patients, more than one transplant may be recommended to adequately control the disease.
Chemotherapeutic agents used for treating the disease are Cyclophosphamid, Doxorubicin, Vincristin and Melphalan, combination therapies with immunomodulating agents such as thalidomide (Thalomid®), lenalidomide (Revlimid®), bortezomib (Velcade®) and corticosteroids (e.g. Dexamethasone) have emerged as important options for the treatment of myeloma, both in newly diagnosed patients and in patients with advanced disease in whom chemotherapy or transplantation have failed.
The currently used therapies are usually not curative. Stem cell transplantation may not be an option for many patients because of advanced age, presence of other serious illness, or other physical limitations. Chemotherapy only partially controls multiple myeloma, it rarely leads to complete remission. Thus, there is an urgent need for new, innovative treatments.
Bellucci et al. (Blood, 2005; 105(10) identified BCMA-specific antibodies in multiple myeloma patients after they had received donor lymphocyte infusions (DLI). Serum of these patients was capable of mediating BCMA-specific cell lysis by ADCC and CDC and was solely detected in patients with anti-tumor responses (4/9), but not in non-responding patients (0/6). The authors speculate that induction of BCMA-specific antibodies contributes to elimination of myeloma cells and long-term remission of patients.
Ryan et al. (Mol. Cancer Ther. 2007; 6(11) reported the generation of an antagonistic BCMA-specific antibody that prevents NF-κB activation which is associated with a potent pro-survival signaling pathway in normal and malignant B-cells. In addition, the antibody conferred potent antibody-dependent cell-mediated cytotoxicity (ADCC) to multiple myeloma cell lines in vitro which was significantly enhanced by Fc-engineering.
Other approaches in fighting blood-borne tumors or autoimmune disorders focus on the interaction between BAFF and APRIL, i.e., ligands of the TNF ligand super family, and their receptors TACI, BAFF-R and BCMA which are activated by BAFF and/or APRIL. For example, by fusing the Fc-domain of human immunoglobulin to TACI, Zymogenetics, Inc. has generated Atacicept (TACI-Ig) to neutralize both these ligands and prevent receptor activation. Atacicept is currently in clinical trials for the treatment of Systemic Lupus Erythematosus (SLE, phase III), multiple sclerosis (MS, phase II) and rheumatoid arthritis (RA, phase II), as well as in phase I clinical trials for the treatment of the B-cell malignancies chronic lymphocytic leukaemia (CLL), non-Hodgkins lymphoma (NHL) and MM. In preclinical studies atacicept reduces growth and survival of primary MM cells and MM cell lines in vitro (Moreaux et al, Blood, 2004, 103) and in vivo (Yaccoby et al, Leukemia, 2008, 22, 406-13), demonstrating the relevance of TACI ligands for MM cells. Since most MM cells and derived cell lines express BCMA and TACI, both receptors might contribute to ligand-mediated growth and survival. These data suggest that antagonizing both BCMA and TACI might be beneficial in the treatment of plasma cell disorders. In addition, BCMA-specific antibodies that cross react with TACI have been described (WO 02/066516).
Human Genome Sciences and GlaxoSmithKline have developed an antibody targeting BAFF which is called Belimumab. Belimumab blocks the binding of soluble BAFF to its receptors BAFF-R, BCMA and TACI on B cells. Belimumab does not bind B cells directly, but by binding BAFF, belimumab inhibits the survival of B cells, including autoreactive B cells, and reduces the differentiation of B cells into immunoglobulin-producing plasma cells.
Nevertheless, despite the fact that BCMA; BAFF-R and TACI, i.e., B cell receptors belonging to the TNF receptor super family, and their ligands BAFF and APRIL are subject to therapies in fighting against cancer and/or autoimmune disorders, there is still a need for having available further options for the treatment of such medical conditions.
Accordingly, there is provided herewith means and methods for the solution of this problem in the form of a binding molecule which is at least bispecific with one binding domain to cytotoxic cells, i.e., cytotoxic T cells, and with a second binding domain to BCMA.
Thus, in a first aspect the present invention provides a binding molecule which is at least bispecific comprising a first and a second binding domain, wherein    (a) the first binding domain is capable of binding to epitope clusters of BCMA as defined herein; and    (b) the second binding domain is capable of binding to the T cell CD3 receptor complex.
A great number of biological pharmaceuticals, such as antibodies and/or other physiologically active proteins, have been produced in the last decade but their efficient production remains a challenging task. In particular, in antibody preparations therapeutically active doses are often in the order of milligram (mg) per administration. Thus, considerable amounts of antibodies are needed as active ingredients. Technologies that allow efficient production of such antibodies, e.g. in heterologous host cells (such as E. coli, yeast or CHO) will lead to cost reduction of antibody preparations and promise a cheap and moreover stable supply to patients.
During the purification of therapeutic antibodies, impurities such as host cell proteins, host cell DNA, process related contaminants (e.g. endotoxins or viral particles) and product variants must be removed. The purification techniques used must be efficient, cost-effective, reliable, and meet the rigorous purity requirements of the final product. Current purification techniques typically involve multiple chromatographic separations. Chromatography techniques exploit inter alia the chemical and physical properties of proteins to achieve a high degree of purification. These chemical and physical properties include for example the size, isoelectric point, charge distribution, hydrophobic sites etc. The various separation modes of chromatography include: ion-exchange, chromatofocussing, gel filtration (size exclusion), hydrophobic interaction, reverse phase, and affinity chromatography. Ion-exchange chromatography (IEX), including anion-exchange and cation-exchange chromatography separates analytes (e.g. therapeutic proteins) by differences of their net surface charges. IEX is a primary tool for the separation of expressed proteins from cellular debris and other impurities. Today, IEX is one of the most frequently used techniques for purification of proteins, peptides, nucleic acids and other charged biomolecules, offering high resolution and group separations with high loading capacity. The technique is capable of separating molecular species that have only minor differences in their charge properties, for example two proteins differing by one charged amino acid. These features make IEX well suited for capture, intermediate purification or polishing steps in a purification protocol and the technique is used from microscale purification and analysis through to purification of kilograms of product. Ion exchange chromatography separates proteins according to their net charge (which is dependent on the composition of the mobile phase). By adjusting the pH or the ionic concentration of the mobile phase, various protein molecules can be separated. This presupposes, however, that the protein in question (the therapeutic antibody for example) has to tolerate the mentioned changes in the pH or the ionic strength during the IEX-procedure. From this it follows that the more tolerant an antibody behaves at un-physiological pH which is required to run an IEX, the more antibody can be recovered/eluted from the IEX column relative to the total amount of loaded protein.
As mentioned before, BCMA is subject to therapies in fighting against cancer and/or autoimmune disorders, but there is still a need for having available further options for the treatment of such medical conditions that can be produced and purified efficiently.
Accordingly, there is provided herewith means and methods also for the solution of the latter technical problem in the form of binding molecules that are disclosed herein.
Thus, in a further aspect, the present invention relates to a binding molecule which is at least bispecific comprising a first and a second binding domain, wherein    (a) the first binding domain is capable of binding to epitope clusters of BCMA as defined herein; and    (b) the second binding domain is capable of binding to the T cell CD3 receptor complex,and wherein the binding molecule is characterized by a percentage of the main peak AUC (Area Under the Curve) of ≥50% relative to the total area of all peaks recovered from a cation exchange column at pH 5.5. In a preferred embodiment, said binding molecule is characterized by a percentage of the main peak AUC (Area Under the Curve) of ≥50% relative to the total area of all peaks recovered from a cation exchange column at pH 5.5 in the following experimental setting:(i) equilibrate a 1 ml cation exchange column with sulphpropyl groups coupled to solid beads with a buffer consisting of 20 mM sodium dihydrogen phosphate and 30 mM sodium chloride adjusted with sodium hydroxide to a pH of 5.5;(ii) dilute 50 μg of binding molecule (preferably monomeric) with dilution buffer consisting of 20 mM sodium dihydrogen phosphate and 30 mM sodium chloride adjusted with sodium hydroxide to a pH of 5.5 to 50 ml final volume;(iii) apply 40 ml of the diluted protein solution to the column followed by an optional wash step with a buffer consisting of 20 mM sodium dihydrogen phosphate and 30 mM sodium chloride adjusted with sodium hydroxide to a pH of 5.5;(iv) elute with a buffer consisting of 20 mM NaH2PO4 and 1000 mM NaCl adjusted with sodium hydroxide to a pH of 5.5 (elution is preferably carried out by a steadily increasing gradient with elution buffer from zero to 100% over a total volume corresponding to 200 column volumes).
Said method is exemplified in the following:
A 1 ml BioPro SP column manufactured by YMC (YMC Europe GmbH, Dinslaken-Germany) with sulphpropyl groups coupled to solid beads was connected to a Äkta Micro FPLC (GE Healthcare) device. For column equilibration, sample dilution and washing a buffer consisting of 20 mM sodium dihydrogen phosphate and 30 mM sodium chloride adjusted with sodium hydroxide to a pH of 5.5 was used.
For elution a buffer consisting of 20 mM NaH2PO4 and 1000 mM NaCl adjusted with sodium hydroxide to a pH of 5.5 was used.
50 μg of BCMA/CD3 bispecific antibody monomer were diluted with dilution buffer to 50 ml final volume and transferred to a GE Amersham Superloop with the same volume.
After column equilibration 40 ml of the diluted protein solution was applied to the column followed by a wash step.
Elution was carried out by a steadily increasing gradient with elution buffer from zero to 100% over a total volume corresponding to 200 column volumes. The whole run was monitored at 280 (blue line) and 254 nm (red line) optical absorption.
For calculation of protein recovery from the column matrix an amount of 50 μg monomeric BCMA/CD3 bispecific antibody was applied to the Äkta Micro Device without an installed column. The resulting peak area was determined and used as 100% reference for comparison with the peak areas measured in the analytical runs.
When applying the above IEX method, the following results could be obtained for 14 representative binding molecules of the present invention.
BCMA/CD3 bispecificAUC of Main PeakRecoveryantibody[%][%]BCMA-546749.4BCMA-535249.4BCMA-838970.2BCMA-628967.0BCMA-59265.4BCMA-989373.4BCMA-718970.2BCMA-349171.8BCMA-747965.4BCMA-208371.8BCMA-908676.6BCMA-918459.0BCMA-309176.6BCMA-509278.1
Thus, in a preferred embodiment, said binding molecule comprises:    (a) (i) the CDRs as defined in SEQ ID Nos 41 to 46, or (ii) the VH and/or VL as defined in SEQ ID Nos 47 and 48, or (iii) the scFv as defined in SEQ ID No 49, or the bispecific BCMA/CD3 binder as defined in SEQ ID No 50;    (b) (i) the CDRs as defined in SEQ ID Nos 191 to 196, or (ii) the VH and/or VL as defined in SEQ ID Nos 197 and 198, or (iii) the scFv as defined in SEQ ID No 199, or the bispecific BCMA/CD3 binder as defined in SEQ ID No 200;    (c) (i) the CDRs as defined in SEQ ID Nos 291 to 296, or (ii) the VH and/or VL as defined in SEQ ID Nos 297 and 298, or (iii) the scFv as defined in SEQ ID No 299, or the bispecific BCMA/CD3 binder as defined in SEQ ID No 300;    (d) (i) the CDRs as defined in SEQ ID Nos 331 to 336, or (ii) the VH and/or VL as defined in SEQ ID Nos 337 and 338, or (iii) the scFv as defined in SEQ ID No 339, or the bispecific BCMA/CD3 binder as defined in SEQ ID No 340;    (e) (i) the CDRs as defined in SEQ ID Nos 491 to 496, or (ii) the VH and/or VL as defined in SEQ ID Nos 497 and 498, or (iii) the scFv as defined in SEQ ID No 499, or the bispecific BCMA/CD3 binder as defined in SEQ ID No 500;    (f) (i) the CDRs as defined in SEQ ID Nos 611 to 616, or (ii) the VH and/or VL as defined in SEQ ID Nos 617 and 618, or (iii) the scFv as defined in SEQ ID No 619, or the bispecific BCMA/CD3 binder as defined in SEQ ID No 620;    (g) (i) the CDRs as defined in SEQ ID Nos 701 to 706, or (ii) the VH and/or VL as defined in SEQ ID Nos 707 and 708, or (iii) the scFv as defined in SEQ ID No 709, or the bispecific BCMA/CD3 binder as defined in SEQ ID No 710;    (h) (i) the CDRs as defined in SEQ ID Nos 731 to 736, or (ii) the VH and/or VL as defined in SEQ ID Nos 737 and 738, or (iii) the scFv as defined in SEQ ID No 739, or the bispecific BCMA/CD3 binder as defined in SEQ ID No 740;    (J) (i) the CDRs as defined in SEQ ID Nos 821 to 826, or (ii) the VH and/or VL as defined in SEQ ID Nos 827 and 828, or (iii) the scFv as defined in SEQ ID No 829, or the bispecific BCMA/CD3 binder as defined in SEQ ID No 830;    (k) (i) the CDRs as defined in SEQ ID Nos 891 to 896, or (ii) the VH and/or VL as defined in SEQ ID Nos 897 and 898, or (iii) the scFv as defined in SEQ ID No 899, or the bispecific BCMA/CD3 binder as defined in SEQ ID No 900;    (l) (i) the CDRs as defined in SEQ ID Nos 901 to 906, or (ii) the VH and/or VL as defined in SEQ ID Nos 907 and 908, or (iii) the scFv as defined in SEQ ID No 909, or the bispecific BCMA/CD3 binder as defined in SEQ ID No 910; and/or    (m) (i) the CDRs as defined in SEQ ID Nos 971 to 976, or (ii) the VH and/or VL as defined in SEQ ID Nos 977 and 978, or (iii) the scFv as defined in SEQ ID No 979, or the bispecific BCMA/CD3 binder as defined in SEQ ID No 980.
In a particular preferred embodiment, said binding molecule comprises or consists of a polypeptide as represented by SEQ ID No: 50, 200, 300, 340, 500, 620, 710, 740, 830, 900, 910 or 980.
In a further preferred embodiment, said binding molecule further comprises at least one protein purification tag such as a GST tag, a FLAG tag, a polyhistidine tag etc. Polyhistidine-tags are preferred. A polyhistidine-tag is typically five or six histidine residues in length, but may be longer. Said protein purification tag may be at the carboxy or amino terminus of the binding molecule. Binding molecules of the present invention, thereby including all specific binding molecules as disclosed herein (e.g. in form of SEQ ID Nos) comprise in a preferred embodiment at least one His-tag that comprises or consists of six histidine residues in length. It is even more preferred that said His-tag is six histidine residues in length and is located at the C-terminus of the binding molecules of the present invention. Thus, in a particularly preferred embodiment of the present invention, said binding molecule of the invention comprises or consists of a polypeptide as represented by SEQ ID No: 50, 200, 300, 340, 500, 620, 710, 740, 830, 900, 910 or 980 and additionally of a hexa-histidine-tag (HHHHHH; SEQ ID NO: 1094) which is located at its C-terminus. It is also preferred that the protein purification tag (His-tag being more preferred and Hexa-His-tag being most preferred) is linked to the C-terminus of the binding molecule of the present invention (preferably consisting of or comprising SEQ ID No: 50, 200, 300, 340, 500, 620, 710, 740, 830, 900, 910 or 980) via a peptide bond.
In a less preferred embodiment, said binding molecule of the present invention comprises or consists of SEQ ID No: 540 or 530 and additionally of a hexa-histidine-tag (HHHHHH; SEQ ID NO: 1094) which is located at its C-terminus and is linked to it via a peptide bond.
In a further preferred embodiment, said binding molecule including the above mentioned protein purification tag(s), His-tags being preferred and Hexa-His-tags at the C-terminus being more preferred, is produced in a host cell as defined herein. CHO is thereby a particularly preferred host cell. It is also preferred that the binding molecules of the invention are characterized by a % dimer conversion after (i) 7 days in solution at 100 μg/ml which is below 2.9, or (ii) 7 days in solution at 250 μg/ml which is below 3.5.
It must be noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.
Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.
The term “and/or” wherever used herein includes the meaning of “and”, “or” and “all or any other combination of the elements connected by said term”.
The term “about” or “approximately” as used herein means within ±20%, preferably within ±15%, more preferably within ±10%, and most preferably within ±5% of a given value or range. Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”.
When used herein “consisting of” excludes any element, step, or ingredient not specified in the claim element. When used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.
In each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms.